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Projects / Programmes source: ARIS

PREPARATION OF SUPPORTED LIPID MEMBRANES WITH ENZYMES FOR DEVELOPMENT OF BIOSENSORS

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Research activity

Code Science Field Subfield
1.02.07  Natural sciences and mathematics  Physics  Biophysics 

Code Science Field
P250  Natural sciences and mathematics  Condensed matter: structure, thermal and mechanical properties, crystallography, phase equilibria 
P260  Natural sciences and mathematics  Condensed matter: electronic structure, electrical, magnetic and optical properties, supraconductors, magnetic resonance, relaxation, spectroscopy 
P300  Natural sciences and mathematics  Analytical chemistry 
P340  Natural sciences and mathematics  Lipids, steroids, membranes 
B120  Biomedical sciences  Molecular biophysics 
T490  Technological sciences  Biotechnology 
Keywords
lateral lipid membrane heterogeneity, cholesterol, supported membranes, membrane-bound proteins, lipid-protein interactions, biosensors, acetylcholinesterase
Evaluation (rules)
source: COBISS
Evaluation of bibliographic research performance indicators according to ARIS methodology
Citations Citations for bibliographic records in COBIB.SI that are linked to records in citation databases
source: WoS source: Scopus source: COBISS
Researchers (1)
no. Code Name and surname Research area Role Period No. of publications
1.  20208  PhD Zoran Arsov  Biotechnology  Head  2007 - 2008  135 
Organisations (1)
no. Code Research organisation City Registration number No. of publications
1.  0106  Jožef Stefan Institute  Ljubljana  5051606000  90,812 
Abstract
Deposition of lipid vesicles on solid supports is an attractive and a simple way of preparing supported lipid membranes. We can expect that the lateral heterogeneity of lipid membranes will influence the formation of supported membranes. Namely, vesicle fusion, which is a fundamental process in the formation of supported lipid bilayers, depends on the membrane structure of lipid vesicles. Lateral heterogeneity is present in both biological and model membranes. In biological membranes lipid and protein molecules organize themselves into lateral domains, while in the case of model membranes phase separation occurs for appropriate composition and temperature. By deposition of membrane vesicles it is also possible to prepare supported membranes with included proteins, which can serve as a biological sensing element in biosensors. The immobilization of membrane-bound proteins through supported membranes avoids the necessity of protein isolation, purification, and reconstitution, which can lead to denaturation or deactivation. Currently the development of biosensors is limited mostly to the use of water-soluble proteins, whereas the application of supported membranes could also boost the use of membrane-bound proteins. The main goal of the proposed project is to establish suitable conditions for preparation of supported lipid membranes by vesicle deposition with respect to the observed lateral heterogeneity in these vesicles. In order to demonstrate an importance of supported membrane formation for development of biosensors based on membrane-bound proteins, supported erythrocyte membrane containing enzyme acetylcholinesterase will be prepared and the function of this enzyme will be checked. Potential application of biosensors based on the enzyme acetylcholinesterase include monitoring of food and environmental samples for the presence of organophosphorus pesticides and insecticides as well as public safety and military/antiterrorism, while such biosensors can be used for the detection of nerve agents.
Significance for science
Results of the project will contribute to the understanding of formation and of possible use of supported lipid membranes without proteins as model systems, or of supported membranes with included proteins in the development of biofunctionalized surfaces. First of all, it is important to understand the role of lateral heterogeneity of lipid membranes in the self-assembly processes affecting the formation of supported membranes by vesicle deposition, such as vesicle fusion or vesicle rupture. Two important steps forward have been made in the study of membrane heterogeneity. Firstly, we have shown that by development of special algorithms for automated analysis of experimental electron paramagnetic resonance (EPR) spectra the efficiency of the extraction of information about membrane structure can be increased. Secondly, we have used the advantage of the attenuated total reflection infrared spectroscopy (ATR-FTIR method) with respect to the conventional transmission method to conduct measurements on supported membranes in excess water. This lead to new findings, while up until now the majority of experiments have been conducted only on membranes hydrated in humid atmosphere. We were also able to demonstrate the advantages of simultaneous use of both methods on equal samples. The results of this part of the project contribute to the knowledge about the phase diagrams for model lipid membranes. These phase diagrams are important, while by changing the composition of membranes we can influence the fraction of particular lipid phase. The development of the described methodology is also very important for research of lateral structure of biological membranes. We would especially like to emphasize the results of the study of intermolecular interactions with ATR-FTIR. We have shown that there exists a direct interaction between cholesterol and carbonyl or amide group through the formation of hydrogen bonds. This is one of the rare experimental evidences of the existence of such interaction. These results shed light on one of the biggest problems in membrane biophysics, which is the problem of stabilization of lateral membrane structure. It has been confirmed with atomic force microscopy (AFM) that the membrane composition affects properties of supported membranes. Therefore, if we determine the properties of some membrane vesicles, we can predict their behavior in the formation of supported membranes. Moreover, we have shown the capability of the scanning near-field infrared microscopy (SNIM) for topographic and chemical characterization of supported membranes, which can be regarded as pioneering steps in the use of this method for the study of supported membranes. Also contribution in ascertaining the possibility of preparation of modified solid supports for formation of nanostructured supported membranes is important. The findings, which can be extracted from our work, have an outstanding significance for application of supported membranes for development of biosensors. In the framework of the project we have also shown the manner in which supported membranes with included membrane-bound proteins or enzymes can be prepared. It is possible to use such functionalized surfaces as biological sensing elements in biosensors. We were able to follow the formation of erythrocyte membranes with included enzyme acetylcholinesterase (AChE) with surface plasmon resonance. The significance of erythrocyte AChE is in its similarity to AChE, which can be found in nerve synapses. This part of the results is very important in the field of development of biosensors based on membrane proteins, while currently the development of biosensors is mostly limited to the use of water-soluble proteins.
Significance for the country
Results of the project will contribute to the understanding of the influence of membrane structure on the formation of supported membranes. The understanding and knowledge about the procedures for preparation of supported membranes with included proteins will enable application of membrane-bound proteins in the development of biosensors and with that the achievement of different socio-economic goals. One of these goals is technological progress, while the development of biosensors is relevant for high-tech production of measurement and diagnostic devices. One example of such devices is biosensor for the detection of blood sugar level in the diabetic patients, which represent a commercial success in the world. Biosensors based on the enzyme acetylcholinesterase (AChE) are important in the following fields: control and care of environment, human health care, security and defense. The reason for this is that such biosensors are able to detect the presence of poisonous and dangerous substances such as pesticides, insecticides, and nerve gases in water, food and environment. The use of biosensors based on AChE would raise the quality of life, while it is for example possible to control the food quality, which is one of the priorities of Slovenian research policy. Results concerning possible modifications of solid supports for preparation of nanostructured supported membranes are important in the field of nanomaterial development. Lately, methods for patterning samples on surfaces on micro- or nanoscale show a significant progress. A controlled deposition of different vesicles with different enzymes on structured and modified substrates could lead to the formation of structured supported membranes. Such structured materials could become a part of concerted action toward development of portable multianalyte and multienzyme biosensors. Connections with various institutions in Slovenia have been established during the project, which have contributed to the transfer of knowledge. For example, commercially available golden supports were used to prepare supported erythrocyte membranes with AChE and to follow their formation by surface plasmon resonance in collaboration with Department of Biology, Biotechnical Faculty, University of Ljubljana. In addition, project activities have been presented in the Laboratory for Environmental Research, University of Nova Gorica, and at the University Institute of Clinical Chemistry and Biochemistry, University Medical Center Ljubljana. The main aim of the presentation was to convey experience of the used methodologies and to search for possibilities of collaboration. During the project we have worked together with young researchers from different research areas, while the project was strongly interdisciplinary. Consequently, we have in indirect way influenced the level of professional capabilities of young experts, which in the future will contribute to the expert systems in health service, in higher education, in research institutes, in pharmaceutical companies and in high-tech companies in the area of biotechnology. With this the project could have an impact on the sustainable development of Slovenian society. A very important part of the project has also been collaboration with groups at the international institutions: Synchrotron Trieste, Italy, Institute of Biophysics and Nanosystems Research of the Austrian Academy of Sciences, Graz, Austria, and Ruhr-University Bochum, Germany. The joint work enabled acquisition of new practical knowledge and the transfer of knowledge to Slovenia.
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